Maximizing Successful Implantation: TLI, PGS and ANXA‐V M2 Haplotype

SARG – Tel AvivMay 2018

Simon FishelCARE Fertility Group

The Embryo – what we know, and don’t

• Aneuploidy testing• Time‐Lapse Imaging

• Morphogenic anomalies• Morphokinetic 

algorithms• Metabolic (‘quiet embryo’) 

assessment• Gene anomalies

Ploidy&TLI

TLI – computing morphokinetics

VP= t1 ‐ tPNfCC1=t2 ‐ t2PBCC2= t4 ‐ t2CC3= t8 ‐ t4CC4= t16 – t8S2= t4 ‐ t3S3= t8 ‐ t5

Compaction= tM ‐ tSCBlastulation= tHN – tSBCollapse= tBCend(n)‐ tBCi(n)

Pribenszky et al 2017: Time‐Lapse Meta‐Analysis 

– Meta‐analysis of RCT of morphokinetic algorithms v single time point morphology for embryo selection

– Outcome measures:• Clinical pregnancy, Live birth, EPL, Stillbirth

– 5 RCTs (n=1637)• LB: 44.2% v 31.3% (OR= 1.668; P<0.009)• EPL: 15.3% v 21.3% (OR= 0.662; P<0.019)• Stillbirth: NS

“time‐lapse is shown to significantly improve overall clinical outcome”

TLI – computing morphokinetics: ‘CAREmaps’

VP= t1 ‐ tPNfCC1=t2 ‐ t2PBCC2= t4 ‐ t2CC3= t8 ‐ t4CC4= t16 – t8S2= t4 ‐ t3S3= t8 ‐ t5

Compaction= tM ‐ tSCBlastulation= tHN – tSBCollapse= tBCend(n)‐ tBCi(n)

TLI - QA

Patient N F 154104 well 3 D4

Embryologist PB2

Pna

2PN PNf

2 cell

3 cell

4 cell

5 cell 6c 7c 8c 9+

st comp

morula TSB TB EB HB

CC2

Abi 2.4 7.612.2

24.1 26 39.4 39.6

52.8

54.4

55.6

55.9

71.7 76.3 94.9

13.4

Cath 2.4 9.414.7

24.1 26 39.4 39.6

52.8

54.4

55.6

55.9 72 85.7

13.4

Claire 2.5 6.723.6

23.9 26 39.4 39.6

52.8

54.6

55.6

56.3

71.7 76 93.9

13.4

Rachel 2.9 7.614.1

24.3

26.2 39.4 39.7

52.9

54.6

55.6

55.9

71.8 76.5 92.4

94.4

13.2

Sarah 2.9 9.210.7

23.9

26.2 39.4 39.7

52.9

54.6

55.8

56.1

71.7 79.2 94.3

13.2

Median 2.5 7.614.1

24.1 26 39.4 39.6

52.8

54.6

55.6

55.9

71.7 76.5

13.4

Mean 2.6 8.115.1

24.1

26.1 39.4 39.6

52.8

54.5

55.6 56

71.8 78.7 93.9

13.3

Max 2.9 9.423.6

24.3

26.2 39.4 39.7

52.9

54.6

55.8

56.3

72.0 85.7 94.9

13.4

Min 2.4 6.710.7

23.9 26 39.4 39.6

52.8

54.4

55.6

55.9

71.7 76 92.4

13.2

EmbryoScope

QA – Attainment Scores

CARE Fertility Group – 1000 Live Births

Live births = 973 deliveries Mean ♀ Age: CAREmaps =  36.9 

v SI = 35.3

Inclusive Confounding Variables

• Embryoscope (y/n)• Patient age (<38/38+)• Day of embryo transfer• No. embryos transferred• Patient type • Donor age • Total previous cycles• Total no. previous live births• ICSI (y/n)• Total no. miscarriages• Intralipid (y/n)• Duration of infertility

• Total # ectopic• BMI • AMH • AFC • Gonadotropin type• Gonadotropin dosing days• Gonadotropin total dose• # Eggs collected• # M2 eggs• Ratio M2 eggs to total• # M2 eggs fertilised

Confounding variables included in the model

• Embryoscope (y/n)• Patient age (<38/38+)• Day of embryo transfer• No. embryos transferred• Patient type • Donor age • Total previous cycles• Total no. previous live births• ICSI (y/n)• Total no. miscarriages• Intralipid (y/n)• Duration of infertility

• Total # ectopic• BMI • AMH • AFC • Gonadotropin type• Gonadotropin dosing days• Gonadotropin total dose• # Eggs collected• # M2 eggs• Ratio M2 eggs to total• # M2 eggs fertilised

CARE Fertility Data (Live Birth Outcome Only)

24,000 records of treatment

• 21,379 Standard incubation treatments

• 2,527 Embryoscope treatments

• 14,878 unique patientsStatistical Analysis:*multiple variable logistic regression models were fit to assess the effects of embryo rank on each potential confounder* Akaike information criterion (AIC) penalty fit for number of parameters and stepwise selection

Live Births (delivery events age <38) – Sig Points

0.0

10.0

20.0

30.0

40.0

50.0

60.0

# pos Beta # with >=1FH Biochem Loss # Implantations # Clinmiscarriage

# Live Birth Event babies/EmbTrd

%

* P<0.0001

* P<0.0001* P<0.0001

* P<0.0001 * P<0.001

TLI   SI

LIVE BIRTH OUTCOME – all ages(CAREmaps 1,007 Deliveries v Standard 6,874 Deliveries)

0

5

10

15

20

25

30

35

40

45

Overall Single Blast

Uplift

* *

* *

* P<0.0001

19%

25%

Live Births (delivery events)

CAREmaps   SI Significance diluted in >37 age group

CAREmaps – Hierarchical Ranking of Embryos

2nd Study in the ‘Trilogy’ –Rbmonline – 2018, in press 

Time‐Lapse Imaging Algorithms Rank Human Preimplantation Embryos According to their Probability to Result in a Live Birth. 

Simon Fishel1a*, Alison Campbella, Sue Montgomeryb, Rachel Smithc, Lynne Niced, Samantha Duffyb, Lucy Jennere, Kathryn Berrisford, Louise Kellame, Rob Smithf, Fiona Foadg, Ashley Becclesa

Hierarchical selection of embryos:

# ET%

LBRMiscarriage

(%)A 373 51.7 24.0B 297 35.0 32.5C 93 31.2 32.6D 80 13.8 35.3

0.0

10.0

20.0

30.0

40.0

50.0

60.0

A B C X

% LBRMiscarriage (%)

47.7%

A tSB or relSBIVF ≤ 93.1hB tSB or relSBIVF >93.1 h

dB ≤ 12.5 hC tSB or relSBIVF >93.1 h

dB >12.5 hD Unable to be

annotated

Single Blast Transfer

D

P<0.001

Ranked embryo LB outcome. 

• D << A (OR = 0.3046; P<0.010)• D << B (OR = 0.428; P<0.01)• B < A   (OR = 0.7114; P<0.01)• C <A    (OR = 0.6501; P< 0.01) • B > C   (OR = 1.09; P<0.01) • C >> D (OR=2.135; p<0.01)

Strong evidence of an effect of embryo rank on the odds of live 

births. 

A has a 233% chance of LB compared to D

CAREmaps v morphology: 

Grade 2:2 highest LB!

1:1/1:2/2:1 2:2/2:3/3:2/3:3

Odd

s Ratio com

pared to transfer of 2

:2

CAREmaps Morphokinetics v Morphology

1373  Blast SET cycles679 (50%) Live Births

0

10

20

30

40

50

60

70

AB

CD

%

Grade

Live Births (%) v MK Grade

0

10

20

30

40

50

60

1:1 1:2 2:1 2:2 2:3 3:3

Morphology

Live Births (%) v Morphology

CAREmaps Morphokinetics v Morphology

Strong evidence of the superiority of the embryo rank model compared with the transfer grade model

(p<0.0001)

TLI – Cleavage anomalies

BMA Annual Book Awards 2016

‘Reverse Cleavage’

PGT‐A

Meta-analyses

01234567

Ris

k R

atio

Observational Studies

PGS vs. Routine Care Clinical IR (Gestational Sacs/#ET)

Matched Cohort Studies of PGS in AMA WomenClinical Pregnancy Rate (IR)

Favo

rs P

GS

Favors Control Clinical IR 1.78x more likely with PGS in

observational studies

US CDC –SART data 2014  ‐ Embryo ‘Success’

# Embryos Transferred 139,832# babies born from those embryos 35,631

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

<35 35‐37 38‐40 41‐42 43‐44 >44 TOTAL

Non‐PGS %

PGS %

12%; ≡16,780

STAR Trial - Illumina

ASRM - 2107

Sig ↑ in CPR/LB if ♀ >35 years

50.8% in the PGS arm vs 37.2% in the control arm

36.5%

0

10

20

30

40

50

60

PGS Non-PGS

CP/ET with or without PGS

% L

IVE

BIRT

H

X2 = 23.0615. P=.00000252%

38%

CARE Fertility Group Data

♀ Age >35n = 365

Annexin A5 (ANXA5) M2 Haplotype (Stratification/PBM)

ANXA 5 M2 haplotype is 4 base pair mutation in the ANXA5

gene causing a reduction in Annexin 5 - normally present in

high concentration on the apical surface of

syncytiotrophoblasts

It’s presence indicates risk => hypercoagulation & and

thrombotic risk

Studies demonstrating association with pregnancy outcome complications & benefit of anti thrombotic therapy

n = 29

Reviews of Annexin A5

Bouter et al; (2015) Placenta 36, Supplement 1, Trophoblast Research, Vol. 29: S43–S49

Degrelle et al; (2017) Sci. Rep. 7, 3

ANXA5 M2 - Haplotype

Annexin A5 gene variantsInitially found through systematic sequence analysis of the ANXA5 gene in 70 patients in Northern German origin with RPL (>2 foetal losses), non-carriers of PTm and/or FVL mutations. To Date: * >4,500 patients of * Different ethnicities and various obstetric complications have been genotypedAssociated risk in Anxa5 M2 Haplotype carriers is 2.4-4.0 for the various thrombophilia-related phenotypes

Annexin-V M2 characteristics

• Defect conveyed embryonally• Risk is independent of maternal / paternal transmission• Distinct from other thrombotic disorders• Spectrum of pathologies as the lesion is in the placenta

PMPC (placenta-mediated pregnancy complications)

CARE: Incidence in our IVF population

Male 26%Female 24%Both partners carriers ~10%Couples 44%Couples w. unexplained infertility 37%Co-exists w. male and female infertility patients (27%) Co exists w PCOS (35%)

Rbmonline 29; 80–87: 2014

M2 Haplotype positive – treatment results

doi:10.1016/j.ebiom.2016.06.024 (2016)

M2 Positive M2 Positive

CARE strategy to maximising LBR

CAREmaps for all patients ♀age <38 = 19-25% ↑ Live Birth♀age >35 => PGT-A♀age >35: PGT-A and CAREmaps NOT mutually exclusivePersonalised Genetics – AnnexinV M2 Haplotype

CARE fertility group

CARE FERTILITY GROUP OF CLINICS!

www.carefertility.co

Thank you for listening

PGT-A & CAREmaps?

• Defect conveyed embryonally• Risk is independent of maternal / paternal transmission.3

• Spectrum of pathologies as the lesion is in the placenta• M2 haplotype present results in a significant reduction in Annexin 5 in the syncytiotrophoblasts..

Conclusion

To Maximise Live Births

ATTENTION TO DETAIL √CONSISTENCY ?USE OF OBJECTIVE TOOLS ?INCREMENTAL INCREASE

HFEA Latest Results, 2017: CPR/Embryo

CLINICAL PREGANCY RATES <35 CLINICAL PREGANCY RATES All Ages 

Clinic CS Total EmbsTrfd ET ORClinical Preg 

(FH)% per 

EmbsTrfd Clinic CS Total EmbsTrfd ET ORClinical Preg 

(FH) % per EmbsTrfdCARE Nottingham 313 327 279 297 169 51.7% CARE Northampton 529 670 483 520 249 37.2%CARE Northampton 276 310 252 272 151 48.7% CARE Nottingham 656 755 577 621 280 37.1%Centre for Reproductive Medicine, Coventry 363 317 292 323 147 46.4% NURTURE Fertility 681 852 596 651 293 34.4%NURTURE Fertility 351 372 303 335 165 44.4% Centre for Reproductive Medicine, Coventry 649 632 520 580 213 33.7%CARE Manchester 372 415 330 364 183 44.1% CARE Sheffield 372 492 332 358 164 33.3%Hewitt Fertility Centre 1109 960 870 1088 422 44.0% Hewitt Fertility Centre 2103 1989 1649 2033 645 32.4%CREATE, St Paul's London 120 135 112 118 56 41.5% IVI Midland 446 573 386 428 184 32.1%CARE Sheffield 214 244 193 209 101 41.4% Hewitt Fertility Centre, Knutsford 88 78 70 86 25 32.1%Bath Fertility Centre 174 180 158 168 73 40.6% Bourn Hall (Norwich) Limited 408 497 354 380 158 31.8%Nuffield Health Woking Hospital 377 402 330 366 161 40.0% CARE Manchester 876 1002 709 825 317 31.6%Ninewells Hospital 235 205 167 214 82 40.0% Leeds Fertility 1355 1573 1163 1278 495 31.5%Leeds Fertility 671 683 587 634 273 40.0% The Gateshead Fertility Unit 482 527 414 446 164 31.1%

Edinburgh Assisted Conception Unit 213 207 174 209 82 39.6% The Centre for Reproductive and Genetic Health 475 776 453 468 241 31.1%The Gateshead Fertility Unit 268 287 238 253 112 39.0% BMI The Chaucer Hospital 133 188 118 126 57 30.3%

Assisted Reproduction and Gynaecology Centre 187 359 185 187 140 39.0% Ninewells Hospital 465 467 356 428 141 30.2%Hewitt Fertility Centre, Knutsford 41 36 34 41 14 38.9% Glasgow Royal Infirmary 1030 1136 828 952 339 29.8%The Agora Gynaecology and Fertility Centre 217 216 190 204 83 38.4% CARE Tunbridge Wells 538 660 454 486 193 29.2%

The Centre for Reproductive and Genetic Health 157 245 154 155 94 38.4% Sussex Downs Fertility Centre 256 342 231 248 97 28.4%CARE Tunbridge Wells 301 326 252 265 125 38.3% Birmingham Women's Hospital 967 1019 790 911 287 28.2%CARE London 192 200 148 178 76 38.0% Nuffield Health Woking Hospital 987 1228 862 933 342 27.9%BMI The Chaucer Hospital 78 108 69 74 41 38.0% CREATE, St Paul's London 336 444 316 330 123 27.7%Sussex Downs Fertility Centre 124 145 108 120 55 37.9% Edinburgh Assisted Conception Unit 561 616 477 547 170 27.6%The Lister Fertility Clinic 519 612 456 510 232 37.9% The Agora Gynaecology and Fertility Centre 565 655 488 526 179 27.3%

Bourn Hall (Norwich) Limited 222 254 192 209 94 37.0% Assisted Reproduction and Gynaecology Centre 543 1170 538 543 316 27.0%Manchester Fertility 209 175 140 197 64 36.6% Barts Health Centre for Reproductive Medicine 991 1128 847 955 296 26.2%Birmingham Women's Hospital 527 517 426 503 187 36.2% Bourn Hall Clinic 1585 2000 1399 1525 516 25.8%Glasgow Royal Infirmary 542 557 428 500 198 35.5% Wolfson Family Clinic 924 1232 844 897 312 25.3%London Women's Clinic 289 273 202 256 96 35.2% St Mary's Hospital 1122 1369 911 1061 342 25.0%IVI Midland 225 294 204 222 102 34.7% Shropshire and Mid‐Wales Fertility Centre 323 418 263 293 104 24.9%King’s Hewitt Fertility Centre 470 473 395 444 162 34.2% King’s Hewitt Fertility Centre 1133 1395 957 1076 347 24.9%Reproductive Genetics Institute 32 59 31 32 20 33.9% Oxford Fertility 1480 1967 1331 1462 489 24.9%Bourn Hall Clinic 794 934 717 774 316 33.8% Hull IVF Unit 413 469 353 391 116 24.7%South West Centre for Reproductive Medicine 142 175 108 123 59 33.7% Wales Fertility Institute‐Neath 346 409 260 312 100 24.4%NewLife Fertility Centre 52 84 50 51 28 33.3% Complete Fertility Centre Southampton 414 442 327 399 108 24.4%London Women's Clinic, Wales 126 147 105 119 49 33.3% Manchester Fertility 518 492 346 466 120 24.4%Wessex Fertility Limited 240 195 173 236 65 33.3% South West Centre for Reproductive Medicine 293 391 229 254 95 24.3%Barts Health Centre for Reproductive Medicine 497 549 427 487 177 32.2% Regional Fertility Centre, Belfast 837 976 683 792 236 24.2%Boston Place 115 149 102 112 47 31.5% BMI The Priory Hospital 447 583 399 430 140 24.0%Glasgow Nuffield Hospital 43 54 36 38 17 31.5% The Lister Fertility Clinic 2039 2594 1677 1949 621 23.9%

Centre for Reproduction & Gynaecology Wales (CRGW) 206 242 163 187 76 31.4% CREATE Centre for Reproduction and Advanced Technology 172 278 160 166 66 23.7%Shropshire and Mid‐Wales Fertility Centre 149 172 123 135 54 31.4% Glasgow Nuffield Hospital 138 189 121 128 44 23.3%Oxford Fertility 749 935 680 746 293 31.3% Bath Fertility Centre 398 498 357 385 114 22.9%Bristol Centre for Reproductive Medicine 319 413 290 297 129 31.2% Leicester Fertility Centre 283 413 258 275 94 22.8%BMI The Priory Hospital 213 269 198 207 83 30.9% London Women's Clinic, Wales 315 380 245 287 86 22.6%Hull IVF Unit 237 253 201 223 78 30.8% Boston Place 426 606 362 398 136 22.4%Cambridge IVF 24 26 21 24 8 30.8% Bourn Hall Clinic (Colchester) 593 706 499 561 158 22.4%

CREATE Centre for Reproduction and Advanced Technology 51 73 47 50 22 30.1% The James Cook University Hospital 281 460 269 275 100 21.7%

Leicester Fertility Centre 165 223 147 160 67 30.0% Assisted Reproduction Unit (ARU), University Hospital of Hartlepool 187 298 166 178 64 21.5%Wolfson Family Clinic 490 608 449 479 181 29.8% GCRM ‐ Belfast 334 490 283 313 105 21.4%Chelsea & Westminster Hospital 144 242 138 144 72 29.8% Newcastle Fertility Centre at Life 672 846 611 643 181 21.4%Complete Fertility Centre Southampton 241 227 188 237 67 29.5% Cambridge IVF 97 118 87 96 25 21.2%Wales Fertility Institute‐Neath 188 225 145 176 66 29.3% Bristol Centre for Reproductive Medicine 844 1328 765 794 280 21.1%St Mary's Hospital 680 807 553 648 231 28.6% NewLife Fertility Centre 171 310 160 164 65 21.0%Herts and Essex Fertility Centre 164 231 151 159 66 28.6% Wessex Fertility Limited 609 556 421 569 116 20.9%Concept Fertility 21 25 18 20 7 28.0% Herts and Essex Fertility Centre 422 657 375 398 135 20.5%Bourn Hall Clinic (Colchester) 325 360 271 310 99 27.5% CARE London 850 965 577 744 198 20.5%Glasgow Centre for Reproductive Medicine 153 211 140 147 58 27.5% Chelsea & Westminster Hospital 438 812 419 434 164 20.2%

Regional Fertility Centre, Belfast 485 567 409 472 155 27.3% Centre for Reproduction & Gynaecology Wales (CRGW) 544 716 417 485 143 20.0%Guys Hospital 742 900 691 726 243 27.0% Jessop Fertility 617 794 507 586 156 19.6%Newcastle Fertility Centre at Life 396 480 364 383 128 26.7% Aberdeen Fertility Centre 478 625 422 443 118 18.9%Jessop Fertility 304 337 244 286 86 25.5% Reproductive Genetics Institute 221 502 213 221 94 18.7%GCRM ‐ Belfast 139 202 120 132 51 25.2% London Women's Clinic 1177 1309 831 973 242 18.5%

Assisted Reproduction Unit (ARU), University Hospital of Hartlepool 137 216 122 132 54 25.0% Glasgow Centre for Reproductive Medicine 531 793 449 493 139 17.5%Brentwood Fertility Centre 23 32 21 21 8 25.0% Homerton Fertility Centre 465 641 389 437 112 17.5%Aberdeen Fertility Centre 241 292 221 233 72 24.7% Guys Hospital 2086 3082 1908 2018 536 17.4%Homerton Fertility Centre 156 191 128 145 47 24.6% Concept Fertility 87 121 70 80 21 17.4%The James Cook University Hospital 183 298 180 181 67 22.5% London Women's Clinic, Darlington 167 171 106 158 29 17.0%Harley Street Fertility Clinic 64 87 50 60 19 21.8% Salisbury Fertility Centre 357 558 307 336 94 16.8%Fertility Exeter 162 207 124 137 45 21.7% Fertility Exeter 338 420 247 285 69 16.4%London Women's Clinic, Darlington 70 66 41 67 14 21.2% Reproductive Health Group 154 122 101 138 20 16.4%BMI Chelsfield Park ACU 84 125 74 81 25 20.0% BMI Chelsfield Park ACU 190 293 162 180 44 15.0%Origin Fertility Care 82 127 72 80 25 19.7% Harley Street Fertility Clinic 162 243 123 147 36 14.8%Salisbury Fertility Centre 203 300 173 191 57 19.0% City Fertility 179 247 143 162 35 14.2%St Jude's Women's Hospital 24 43 23 24 8 18.6% Origin Fertility Care 226 363 190 214 48 13.2%City Fertility 61 80 51 57 14 17.5% London Fertility Centre 407 790 358 403 103 13.0%The Fertility & Gynaecology Academy 18 24 16 18 4 16.7% St Jude's Women's Hospital 73 133 67 73 15 11.3%Reproductive Health Group 47 36 29 42 6 16.7% The Fertility & Gynaecology Academy 96 116 62 89 13 11.2%London Fertility Centre 41 69 36 41 7 10.1% Brentwood Fertility Centre 85 110 65 73 12 10.9%Wales Fertility Institute ‐ Cardiff 117 144 99 107 14 9.7% Wales Fertility Institute ‐ Cardiff 281 340 226 248 30 8.8%

National Average 19695 22545 16828 18852 7524 33.4% National Average 44517 56605 37488 42016 13854 24.5%

Fresh IVF/ICSI Own Eggs: July 2015‐July 2016 Fresh IVF/ICSI Own Eggs: July 2015‐July 2016 

CLINICAL PREGANCY RATES <35 CLINICAL PREGANCY RATES All Ages 

Clinic CSTotal 

EmbsTrfd ET ORClinical Preg (FH)

% per EmbsTrfd Clinic CS

Total EmbsTrfd ET OR

Clinical Preg (FH)

% per EmbsTrfd

CARE Nottingham 313 327 279 297 169 51.7% CARE Northampton 529 670 483 520 249 37.2%

CARE Northampton 276 310 252 272 151 48.7% CARE Nottingham 656 755 577 621 280 37.1%

Nat Ave <35: 33.4%All Ages: 24.5%

♀<35 ♀ All Ages

US CDC SARTAll Ages: 25.5%

Netherlands<18%

For example, the estimated odds ratio for an embryo of grade 32 compared with 11 is 0.1619 with a 95% CI of (0.036, 0.520), indicating that there is strong evidence (Wald p‐value = 0.0056) that embryos of grade 32 are less likely to result in a live birth than embryos of grade 11. 

Grade OR 95% Cl P value

12v11 1.0407 (0.661, 1.637) 0.8631

21v11 1.0248 (0.618, 2.382) 0.5866

22v11 1.0563 (0.733, 1.159) 0.768

23v11 0.3684 (0.215, 0.623) 0.002

32v11 0.1619 (0.036, 0.520) 0.0056

33v11 0.2339 (0.082, 0.582) 0.0032

Descriptive statistics are provided to characterise the covariates available across the embryo ranks and transfer grades for standard patients only. For the binary and categorical variables considered in the analysis, cross‐tabulations versus rank (A/B/C/D) are given in Table 9 and cross‐tabulations versus transfer grade (11/12/21/22/23/32/33) are given in Table 10. For the continuous/discrete variables considered in the analysis, summary statistics split by embryo rank are given in Table 11, and by transfer grade in Table 12. 

showed strong evidence of the superiority of the embryo rank model compared with the transfer grade model (p<0.0001 [raw], p<0.0001 [AIC‐corrected])